EPIC-0628 abrogates HOTAIR/EZH2 interaction and enhances the temozolomide efficacy via promoting ATF3 expression and inhibiting DNA damage repair in glioblastoma.

The efficacy of temozolomide (TMZ) treatment in glioblastoma (GBM) is influenced by various mechanisms, mainly including the level of O6-methylguanine-DNA methyltransferase (MGMT) and the activity of DNA damage repair (DDR) pathways. In our previous study, we had proved that long non-coding RNA HOTAIR regulated the GBM progression and mediated DDR by interacting with EZH2, the catalytic subunit of PRC2. In this study, we developed a small-molecule inhibitor called EPIC-0628 that selectively disrupted the HOTAIR-EZH2 interaction and promoted ATF3 expression. The upregulation of ATF3 inhibited the recruitment of p300, p-p65, p-Stat3 and SP1 to the MGMT promoter. Hence, EPIC-0628 silenced MGMT expression. Besides, EPIC-0628 induced cell cycle arrest by increasing the expression of CDKN1A and impaired DNA double-strand break repair via suppressing the ATF3-p38-E2F1 pathway. Lastly, EPIC-0628 enhanced TMZ efficacy in GBM in vitro and vivo. Hence, this study provided evidence for the combination of epigenetic drugs EPIC-0628 with TMZ for GBM treatment through the above mechanisms.

Mechanism of AGT-Mediated Repair of dG-dC Cross-Links in the Drug Resistance to Chloroethylnitrosoureas: Molecular Docking, MD Simulation, and ONIOM (QM/MM) Investigation.

Chloroethylnitrosoureas (CENUs) are important chemotherapies applied in the treatment of cancer. They exert anticancer activity by inducing DNA interstrand cross-links (ICLs) via the formation of two O6-alkylguanine intermediates, O6-chloroethylguanine (O6-ClEtG) and N1,O6-ethanoguanine (N1,O6-EtG). However, O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair enzyme, can restore the O6-alkylguanine damages and thereby obstruct the formation of ICLs (dG-dC cross-link). In this study, the inhibitory mechanism of ICL formation was investigated to elucidate the drug resistance of CENUs mediated by AGT in detail. Based on the structures of the substrate-enzyme complexes obtained from docking and MD simulations, two ONIOM (QM/MM) models with different sizes of the QM region were constructed. The model with a larger QM region, which included the substrate (O6-ClEtG or N1,O6-EtG), a water molecule, and five residues (Tyr114, Cys145, His146, Lys165, and Glu172) in the active pocket of AGT, accurately described the repairing reaction and generated the results coinciding with the experimental outcomes. The repair process consists of two sequential steps: hydrogen transfer to form a thiolate anion on Cys145 and alkyl transfer from the O6 site of guanine (the rate-limiting step). The repair of N1,O6-EtG was more favorable than that of O6-ClEtG from both kinetics and thermodynamics aspects. Moreover, the comparison of the repairing process with the formation of dG-dC cross-link and the inhibition of AGT by O6-benzylguanine (O6-BG) showed that the presence of AGT could effectively interrupt the formation of ICLs leading to drug resistance, and the inhibition of AGT by O6-BG that was energetically more favorable than the repair of O6-ClEtG could not prevent the repair of N1,O6-EtG. Therefore, it is necessary to completely eliminate AGT activity before CENUs medication to enhance the chemotherapeutic effectiveness. This work provides reasonable explanations for the supposed mechanism of AGT-mediated drug resistance of CENUs and will assist in the development of novel CENU chemotherapies and their medication strategies.

Integration of Demethylation-Activated DNAzyme with a Single Quantum Dot Nanosensor for Sensitive Detection of O6-Methylguanine DNA Methyltransferase in Breast Tissues.

O6-Methylguanine-DNA-methyltransferase (MGMT) is a demethylation protein that dynamically regulates the O6-methylguanine modification (O6 MeG), and dysregulated MGMT is implicated in various malignant tumors. Herein, we integrate demethylation-activated DNAzyme with a single quantum dot nanosensor to sensitively detect MGMT in breast tissues. The presence of MGMT induces the demethylation of the O6 MeG-caged DNAzyme and the restoration of catalytic activity. The activated DNAzyme then specifically cleaves the ribonucleic acid site of hairpin DNA to expose toehold sequences. The liberated toehold sequence may act as a primer to trigger a cyclic exponential amplification reaction for the generation of enormous signal strands that bind with the Cy5/biotin-labeled probes to form sandwich hybrids. The assembly of sandwich hybrids onto 605QD obtains 605QD-dsDNA-Cy5 nanostructures, inducing efficient FRET between the 605QD donor and Cy5 acceptor. Notably, the introduction of a mismatched base in hairpin DNA can greatly minimize the background and improve the signal-to-noise ratio. This nanosensor achieves a dynamic range of 1.0 × 10-8 to 0.1 ng/µL and a detection limit of 155.78 aM, and it can screen MGMT inhibitors and monitor cellular MGMT activity with single-cell sensitivity. Moreover, it can distinguish the MGMT level in tissues of breast cancer patients and healthy persons, holding great potential in clinical diagnostics and epigenetic research studies.

Adoptive cell therapy for high grade gliomas using simultaneous temozolomide and intracranial mgmt-modified γδ t cells following standard post-resection chemotherapy and radiotherapy: current strategy and future directions.

Cellular therapies, including chimeric antigen receptor T cell therapies (CAR-T), while generally successful in hematologic malignancies, face substantial challenges against solid tumors such as glioblastoma (GBM) due to rapid growth, antigen heterogeneity, and inadequate depth of response to cytoreductive and immune therapies, We have previously shown that GBM constitutively express stress associated NKG2D ligands (NKG2DL) recognized by gamma delta (γδ) T cells, a minor lymphocyte subset that innately recognize target molecules via the γδ T cell receptor (TCR), NKG2D, and multiple other mechanisms. Given that NKG2DL expression is often insufficient on GBM cells to elicit a meaningful response to γδ T cell immunotherapy, we then demonstrated that NKG2DL expression can be transiently upregulated by activation of the DNA damage response (DDR) pathway using alkylating agents such as Temozolomide (TMZ). TMZ, however, is also toxic to γδ T cells. Using a p140K/MGMT lentivector, which confers resistance to TMZ by expression of O(6)-methylguanine-DNA-methyltransferase (MGMT), we genetically engineered γδ T cells that maintain full effector function in the presence of therapeutic doses of TMZ. We then validated a therapeutic system that we termed Drug Resistance Immunotherapy (DRI) that combines a standard regimen of TMZ concomitantly with simultaneous intracranial infusion of TMZ-resistant γδ T cells in a first-in-human Phase I clinical trial (NCT04165941). This manuscript will discuss DRI as a rational therapeutic approach to newly diagnosed GBM and the importance of repeated administration of DRI in combination with the standard-of-care Stupp regimen in patients with stable minimal residual disease.

Somatostatin Receptor Subtype-2 Targeting System for Specific Delivery of Temozolomide.

Temozolomide (TMZ) is a DNA alkylating agent that produces objective responses in patients with neuroendocrine tumors (NETs) when the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) is inactivated. At high doses, TMZ therapy exhausts MGMT activity but also produces dose-limiting toxicities. To reduce off-target effects, we converted the clinically approved radiotracer 68Ga-DOTA-TOC into a peptide-drug conjugate (PDC) for targeted delivery of TMZ to somatostatin receptor subtype-2 (SSTR2)-positive tumor cells. We used an integrated radiolabeling strategy for direct quantitative assessment of receptor binding, pharmacokinetics, and tissue biodistribution. In vitro studies revealed selective binding to SSTR2-positive cells with high affinity (5.98 ± 0.96 nmol/L), internalization, receptor-dependent DNA damage, cytotoxicity, and MGMT depletion. Imaging and biodistribution analysis showed preferential accumulation of the PDC in receptor-positive tumors and high renal clearance. This study identified a trackable SSTR2-targeting system for TMZ delivery and utilizes a modular design that could be broadly applied in PDC development.

Fluorescent molecular rotors detect O6-methylguanine dynamics and repair in duplex DNA.

Alkylation at the O6 position of guanine is a common and highly mutagenic form of DNA damage. Direct repair of O6-alkylguanines by the "suicide" enzyme O6-methylguanine DNA methyltransferase (MGMT, AGT, AGAT) maintains genome stability and inhibits carcinogenesis. In this study, a fluorescent analogue of thymidine containing trans-stilbene (tsT) is quenched by O6-methylguanine residues in the opposite strand of DNA by molecular dynamics that propagate through the duplex with as much as ∼9 Šof separation. Increased fluorescence of tsT or the cytosine analogue tsC resulting from MGMT-mediated DNA repair were distinguishable from non-covalent DNA-protein binding following protease digest. To our knowledge, this is the first study utilizing molecular rotor base analogues to detect DNA damage and repair activities in duplex DNA.

Epigenetic downregulation of O6-methylguanine-DNA methyltransferase contributes to chronic hexavalent chromium exposure-caused genotoxic effect and cell transformation.

Hexavalent chromium [Cr(VI)] is a common environmental pollutant and chronic exposure to Cr(VI) causes lung cancer and other types of cancer in humans, although the mechanism of Cr(VI) carcinogenesis remains elusive. Cr(VI) has been considered as a genotoxic carcinogen, but accumulating evidence indicates that Cr(VI) also causes various epigenetic toxic effects that play important roles in Cr(VI) carcinogenesis. However, it is not clear how Cr(VI)-caused epigenetic dysregulations contributes to Cr(VI) carcinogenesis. This study investigates whether Cr(VI) epigenetic toxic effect has an impact on its genotoxic effect. It was found that chronic low dose of Cr(VI) exposure time-dependently down-regulates the expression of a critical DNA damage repair protein O6-methylguanine-DNA methyltransferase (MGMT), leading to the increases of the levels of the highly mutagenic and carcinogenic DNA lesion O6-methylguanine (O6-MeG) in human bronchial epithelial BEAS-2B cells. Moreover, the levels of MGMT and O6-MeG in chronic Cr(VI) exposure-caused human lung cancer tissues are also significantly lower and higher than that in the adjacent normal lung tissues, respectively. It was further determined that chronic low dose of Cr(VI) exposure-transformed BEAS-2B cells display impaired DNA damage repair capacity and a high sensitivity to the toxicity of the alkylating chemotherapeutic drug Temozolomide. In contrast, stably overexpressing MGMT in parental BEAS-2B cells reverses chronic low dose of Cr(VI) exposure-caused DNA damage repair deficiency and significantly reduces cell transformation by Cr(VI). Further mechanistical studies revealed that chronic low dose of Cr(VI) exposure down-regulates MGMT expression through epigenetic mechanisms by increasing DNA methylation and histone H3 repressive modifications. Taken together, these findings suggest that epigenetic down-regulation of a crucial DNA damage repair protein MGMT contributes significantly to the genotoxic effect and cell transformation caused by chronic low dose of Cr(VI) exposure.

A modified nucleoside O6-methyl-2'-deoxyguanosine-5'-triphosphate exhibits anti-glioblastoma activity in a caspase-independent manner.

Resistance to temozolomide (TMZ), the frontline chemotherapeutic agent for glioblastoma (GBM), has emerged as a formidable obstacle, underscoring the imperative to identify alternative therapeutic strategies to improve patient outcomes. In this study, we comprehensively evaluated a novel agent, O6-methyl-2'-deoxyguanosine-5'-triphosphate (O6-methyl-dGTP) for its anti-GBM activity both in vitro and in vivo. Notably, O6-methyl-dGTP exhibited pronounced cytotoxicity against GBM cells, including those resistant to TMZ and overexpressing O6-methylguanine-DNA methyltransferase (MGMT). Mechanistic investigations revealed that O6-methyl-dGTP could be incorporated into genomic DNA, disrupting nucleotide pools balance, and inducing replication stress, resulting in S-phase arrest and DNA damage. The compound exerted its anti-tumor properties through the activation of AIF-mediated apoptosis and the parthanatos pathway. In vivo studies using U251 and Ln229 cell xenografts supported the robust tumor-inhibitory capacity of O6-methyl-dGTP. In an orthotopic transplantation model with U87MG cells, O6-methyl-dGTP showcased marginally superior tumor-suppressive activity compared to TMZ. In summary, our research, for the first time, underscores the potential of O6-methyl-dGTP as an effective candidate against GBM, laying a robust scientific groundwork for its potential clinical adoption in GBM treatment regimens.

Base flipping mechanism and binding strength of methyl-damaged DNA during the interaction with AGT.

Methyl damage to DNA bases is common in the cell nucleus. O6-alkylguanine-DNA alkyl transferase (AGT) may be a promising candidate for direct damage reversal in methylated DNA (mDNA) at the O6 point of the guanine. Indeed, atomic-level investigations in the contact region of AGT-DNA complex can provide an in-depth understanding of their binding mechanism, allowing to evaluate the silico-drug nature of AGT and its utility in removing methyl damage in DNA. In this study, molecular dynamics (MD) simulation was utilized to examine the flipping of methylated nucleotide, the binding mechanism between mDNA and AGT, and the comparison of binding strength prior and post methyl transfer to AGT. The study reveals that methylation at the O6 atom of guanine weakens the hydrogen bond (H-bond) between guanine and cytosine, permitting for the flipping of such nucleotide. The formation of a H-bond between the base pair of methylated nucleotide (i.e., cytosine) and the intercalated arginine of AGT also forces the nucleotide to rotate. Following that, electrostatics and van der Waals contacts as well as hydrogen bonding contribute to form the complex of DNA and protein. The stronger binding of AGT with DNA before methyl transfer creates the suitable condition to transfer methyl adduct from DNA to AGT.

Genetic and epigenetic alterations in MGMT gene and correlation with concomitant chemoradiotherapy (CRT) in cervical cancer.

PURPOSE: The MGMT (O6-methylguanine-DNA methyltransferase) gene plays a crucial role in repairing DNA damage caused by alkylating agents, including those used in chemotherapy. Genetic and epigenetic alterations can influence the regulation of MGMT gene, which in turn may impact the response to concomitant chemoradiotherapy (CRT) in cervical cancer. The present study was undertaken to evaluate the correlation of such variations in MGMT gene with the treatment outcome of concomitant chemoradiotherapy (CRT) in cervical cancer. METHODS: A total of 460 study subjects (240 controls and 220 patients) were subjected to genotypic analysis of MGMT gene variants rs12917(T/C) and rs2308327(A/G) by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). Out of them, 48 each of controls and patients were analyzed for promoter methylation and expression by methylation-specific PCR and real-time PCR, respectively. Patients (n = 48) were followed up and evaluated for treatment (CRT) outcome. Statistical analyses were done using GraphPad (9.0) and SPSS version 18.0. RESULTS: Individuals with GG genotype, G allele of rs2308327, and haplotype 'TA' of both variants showed a significant increase in the development of cervical cancer (P ≤ 0.05). In epigenetic regulation, there was a significant hypermethylation of MGMT gene and down-regulation of their expression in patients compared to control individuals. In treatment outcome of CRT, GG genotype of rs2308327(A/G) gene variant showed better response and GG + AG was significantly associated with vital status (alive). Unmethylated MGMT gene showed better median overall survival up to 25 months significant in comparison to methylated MGMT promoter. CONCLUSION: Gene variant rs2308327(A/G) and promoter hypermethylation regulated MGMT gene can be a good prognostic for treatment response in cervical cancer patients.

Modulating MGMT expression through interfering with cell signaling pathways.

Guanine O6-alkylating agents are widely used as first-line chemotherapeutic drugs due to their ability to induce cytotoxic DNA damage. However, a major hurdle in their effectiveness is the emergence of chemoresistance, largely attributed to the DNA repair pathway mediated by O6-methylguanine-DNA methyltransferase (MGMT). MGMT plays an important role in removing the alkyl groups from lethal O6-alkylguanine (O6-AlkylG) adducts formed by chemotherapeutic alkylating agents. By doing so, MGMT enables tumor cells to evade apoptosis and develop drug resistance toward DNA alkylating agents. Although covalent inhibitors of MGMT, such as O6-benzylguanine (O6-BG) and O6-(4-bromothenyl)guanine (O6-4-BTG or lomeguatrib), have been explored in clinical settings, their utility is limited due to severe delayed hematological toxicity observed in most patients when combined with alkylating agents. Therefore, there is an urgent need to identify new targets and unravel the underlying molecular mechanisms and to develop alternative therapeutic strategies that can overcome MGMT-mediated tumor resistance. In this context, the regulation of MGMT expression via interfering the specific cell signaling pathways (e.g., Wnt/ß-catenin, NF-κB, Hedgehog, PI3K/AKT/mTOR, JAK/STAT) emerges as a promising strategy for overcoming tumor resistance, and ultimately enhancing the efficacy of DNA alkylating agents in chemotherapy.

Identification of the E2F1-RAD51AP1 axis as a key factor in MGMT-methylated GBM TMZ resistance.

OBJECTIVE: Epidermal growth factor receptor variant III (EGFRvIII) is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme (GBM). Temozolomide (TMZ) is a standard chemotherapeutic for GBM, but TMZ treatment benefits are compromised by chemoresistance. This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance. METHODS: CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM. Western blot, real-time PCR, flow cytometry, and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1 (RAD51AP1). RESULTS: Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells. Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment. Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells. Knockdown of E2F1 increased sensitivity to TMZ. Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1, mediates TMZ resistance, and has a potential E2F1 binding site on the promoter. Knockdown of RAD51AP1 enhanced the sensitivity of TMZ; however, overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells. Furthermore, RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase (MGMT) expression. The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated, but not MGMT-unmethylated TMZ-treated GBM patients. CONCLUSIONS: Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment. RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair. Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.

EPIC-0307-mediated selective disruption of PRADX-EZH2 interaction and enhancement of temozolomide sensitivity to glioblastoma via inhibiting DNA repair and MGMT.

BACKGROUND: Temozolomide (TMZ) treatment efficacy in glioblastoma (GBM) has been limited by resistance. The level of O-6-methylguanine-DNA methyltransferase (MGMT) and intrinsic DNA damage repair factors are important for the TMZ response in patients. Here, we reported a novel compound, called EPIC-0307, that increased TMZ sensitivity by inhibiting specific DNA damage repair proteins and MGMT expression. METHODS: EPIC-0307 was derived by molecular docking screening. RNA immunoprecipitation (RIP), and chromatin immunoprecipitation by RNA (ChIRP) assays were used to verify the blocking effect. Chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP) assays were performed to explore the mechanism of EPIC-0307. A series of in vivo and in vitro experiments were designed to evaluate the efficacy of EPIC-0307 in sensitizing GBM cells to TMZ. RESULTS: EPIC-0307 selectively disrupted the binding of PRADX to EZH2 and upregulated the expression of P21 and PUMA, leading to cell cycle arrest and apoptosis in GBM cells. EPIC-0307 exhibited a synergistic inhibitory effect on GBM when combined with TMZ by downregulating TMZ-induced DNA damage repair responses and epigenetically silencing MGMT expression through modulating the recruitment of ATF3-pSTAT3-HDAC1 regulatory complex to the MGMT promoter. EPIC-0307 demonstrated significant efficacy in suppressing the tumorigenesis of GBM cells, restoring TMZ sensitivity. CONCLUSION: This study identified a potential small-molecule inhibitor (SMI) EPIC-0307 that selectively disrupted the PRADX-EZH2 interaction to upregulate expressions of tumor suppressor genes, thereby exerting its antitumor effects on GBM cells. EPIC-0307 treatment also increased the chemotherapeutic efficacy of TMZ by epigenetically downregulating DNA repair-associate genes and MGMT expression in GBM cells.

Identifying genetic variants regulating MGMT gene expression - A study in monozygotic Danish twins.

Identifying genetic factors affecting the regulation of the O-6-Methylguanine-DNA Methyltransferase (MGMT) gene and estimating the genetic contribution of the MGMT gene through within-pair correlation in monozygotic twin pairs is of particular importance in various types of cancer such as glioblastoma. We used gene expression data in whole blood from 448 monozygotic twins from the Middle Age Danish Twins (MADT) study to investigate genetic regulation of the MGMT gene by performing a genome-wide association study (GWAS) of the variation in MGMT expression. Additionally, we estimated within-pair dependence measures of the expression values looking for the genetic influence of significant identified genes. We identified 243 single nucleotide polymorphisms (SNPs) significantly (p < 5e-8) associated with expression of MGMT, all located on chromosome 10 near the MGMT gene. Of the 243 SNPs, 7 are novel cis-eQTLs. By further looking into the suggestively significant SNPs (increasing cutoff to p = 1e-6), we identified 11 suggestive trans-eQTLs located on chromosome 17. These variants were in or proximal to a total of seven genes, which may regulate MGMT expression. The within-pair correlation of the expression of MGMT, TRIM37, and SEPT4 provided the upper bound genetic influence of these genes. Overall, identifying cis- or trans-acting genetic variations regulating the MGMT gene can pave the way for a better understanding of the MGMT gene function and ultimately in understanding the patient's sensitivity to therapeutic alkylating agents.

Givinostat Inhibition of Sp1-dependent MGMT Expression Sensitizes Glioma Stem Cells to Temozolomide.

BACKGROUND/AIM: Givinostat is a pan-histone deacetylase (HDAC) inhibitor that has demonstrated excellent tolerability as well as efficacy in patients with polycythemia vera. Accumulating in vitro and in vivo evidence suggests givinostat is also promising as a therapeutic agent targeting glioma stem cells (GSCs), the cancer stem cells of glioblastoma (GBM) considered responsible for its intractable nature. However, it remains to be shown how givinostat impacts the therapeutic effects of temozolomide, a DNA-alkylating agent and the key component of GBM treatment given not only during postoperative radiotherapy but also thereafter as maintenance chemotherapy. MATERIALS AND METHODS: The effects of givinostat and knockdown of O6-methylguanine-DNA methyltransferase (MGMT) or Sp1 on the mRNA and protein expression of relevant genes in human GSC lines were examined by RT-PCR and western blot analyses. The dye exclusion method was used to evaluate cell viability. RESULTS: Givinostat enhanced the cytotoxic activity of temozolomide in GSC lines expressing MGMT, in which the MGMT expression was shown to contribute to their temozolomide resistance. Givinostat inhibited MGMT expression in GSCs and, in parallel, the expression of Sp1, a transcription factor involved in the control of MGMT promoter activity. Knockdown experiments demonstrated Sp1 expression was indeed required for MGMT expression in GSCs. CONCLUSION: Givinostat, in addition to its own cytotoxic activity, sensitizes GSCs to temozolomide by inhibiting Sp1-dependent MGMT expression in GSCs. Combining givinostat with temozolomide could therefore be a rational therapeutic strategy to effectively eliminate GSCs and thus help overcome the therapy resistance of GBM.

The Evolution of SNAP-Tag Labels.

The conjugation of proteins with synthetic molecules can be conducted in many different ways. In this Perspective, we focus on tag-based techniques and specifically on the SNAP-tag technology. The SNAP-tag technology makes use of a fusion protein between a protein of interest and an enzyme tag that enables the actual conjugation reaction. The SNAP-tag is based on the O6-alkylguanine-DNA alkyltransferase (AGT) enzyme and is optimized to react selectively with O6-benzylguanine (BG) substrates. BG-containing dye derivatives have frequently been used to introduce a fluorescent tag to a specific protein. We believe that the site-specific conjugation of polymers to proteins can significantly benefit from the SNAP-tag technology. Especially, polymers synthesized via reversible deactivation radical polymerization allow for the facile introduction of a BG end group to enable SNAP-tag conjugation.

The dual role of DNA repair protein MGMT in cancer prevention and treatment.

Alkylating agents are genotoxic chemicals that can induce and treat various types of cancer. This occurs through covalent bonding with cellular macromolecules, in particular DNA, leading to the loss of functional integrity under the persistence of modifications upon replication. O6-alkylguanine (O6-AlkylG) adducts are proposed to be the most potent DNA lesions induced by alkylating agents. If not repaired correctly, these adducts can result, at the molecular level, in DNA point mutations, chromosome aberrations, recombination, crosslinking, and single- and double-strand breaks (SSB/DSBs). At the cellular level, these lesions can result in malignant transformation, senescence, or cell death. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein capable of removing the alkyl groups from O6-AlkylG adducts in a damage reversal process that can prevent the adverse biological effects of DNA damage caused by guanine O6-alkylation. MGMT can thereby defend normal cells against tumor initiation, however it can also protect tumor cells against the beneficial effects of chemotherapy. Hence, MGMT can play an important role in both the prevention and treatment of cancer; thus, it can be considered as a double-edged sword. From a clinical perspective, MGMT is a therapeutic target, and it is important to explore the rational development of its clinical exploitation.

Iron Oxide Nanoparticles Decorated with Functional Peptides for a Targeted siRNA Delivery to Glioma Cells.

Glioma is a deadly form of brain cancer, and the difficulty of treating glioma is exacerbated by the chemotherapeutic resistance developed in the tumor cells over the time of treatment. siRNA can be used to silence the gene responsible for the increased resistance, and sensitize the glioma cells to drugs. Here, iron oxide nanoparticles functionalized with peptides (NP-CTX-R10) were used to deliver siRNA to silence O6-methylguanine-DNA methyltransferase (MGMT) to sensitize tumor cells to alkylating drug, Temozolomide (TMZ). The NP-CTX-R10 could complex with siRNA through electrostatic interactions and was able to deliver the siRNA to different glioma cells. The targeting ligand chlorotoxin and cell penetrating peptide polyarginine (R10) enhanced the transfection capability of siRNA to a level comparable to commercially available Lipofectamine. The NP-siRNA was able to achieve up to 90% gene silencing. Glioma cells transfected with NP-siRNA targeting MGMT showed significantly elevated sensitivity to TMZ treatment. This nanoparticle formulation demonstrates the ability to protect siRNA from degradation and to efficiently deliver the siRNA to induce therapeutic gene knockdown.

The nanoprodrug of polytemozolomide combines with MGMT siRNA to enhance the effect of temozolomide in glioma.

Temozolomide (TMZ) is a conventional chemotherapeutic drug for glioma, however, its clinical application and efficacy is severely restricted by its drug resistance properties. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme, which can repair the DNA damage caused by TMZ. A large number of clinical data show that reducing the expression of MGMT can enhance the chemotherapeutic efficacy of TMZ. Therefore, in order to improve the resistance of glioma to TMZ, an angiopep-2 (A2) modified nanoprodrug of polytemozolomide (P(TMZ)n) that combines with MGMT siRNA (siMGMT) targeting MGMT was developed (A2/T/D/siMGMT). It not only increased the amount of TMZ within tumor lesion site, but also reduced MGMT expression in glioma. The in vitro experiments indicated that the A2/T/D/siMGMT effectively enhanced the cellular uptake of TMZ and siMGMT, and resulted in a significant cell apoptosis and cytotoxicity in the glioma cells. The in vivo experiments showed that glioma growth was inhibited and the survival time of animals were prolonged remarkably after A2/T/D/siMGMT was injected via tail vein. The results showed that the therapeutic effect of A2/T/D/siMGMT in the treatment of glioma was significantly improved.

Temozolomide Resistance: A Multifarious Review on Mechanisms Beyond O-6-Methylguanine-DNA Methyltransferase.

BACKGROUND: Chemotherapy with the oral alkylating agent temozolomide still prevails as a linchpin in the therapeutic regimen of glioblastoma alongside radiotherapy. Because of the impoverished prognosis and sparse chemotherapeutic medicaments associated with glioblastoma, the burgeoning resistance to temozolomide has made the whole condition almost irremediable. OBJECTIVE: The present review highlights the possible mechanisms of drug resistance following chemotherapy with temozolomide. METHODS: The review summarizes the recent developments, as published in articles from Scopus, PubMed, and Web of Science search engines. DESCRIPTION: One of the prime resistance mediators, O-6-methylguanine-DNA methyltransferase, upon activation, removes temozolomide-induced methyl adducts bound to DNA and reinstates genomic integrity. In the bargain, neoteric advances in the conception of temozolomide resistance have opened the door to explore several potential mediators like indirect DNA repair systems, efflux mechanisms, epigenetic modulation, microenvironmental influences, and autophagy-apoptosis processes that constantly lead to the failure of chemotherapy. CONCLUSION: This review sheds light on recent discoveries, proposed theories, and clinical developments in the field of temozolomide resistance to summarize the complex and intriguing involvement of oncobiological pathways.